Using SiO2-Coated Gold Nanorods as Temperature Jump

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Using SiO-Coated Gold Nanorods as Temperature Jump Photothermal Convertors Coupled with a Confocal Fluorescent Thermometer to Study Protein Unfolding Kinetics: A Case of Bovine Serum Albumin Kuan-Jen Chen, Chia-Te Lin, Ke-Chia Tseng, and Li-Kang Chu J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.7b05033 • Publication Date (Web): 19 Jun 2017 Downloaded from http://pubs.acs.org on June 24, 2017

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The Journal of Physical Chemistry

Using SiO2-Coated Gold Nanorods as Temperature Jump Photothermal Convertors Coupled with a Confocal Fluorescent Thermometer to Study Protein Unfolding Kinetics: A Case of Bovine Serum Albumin Kuan-Jen Chen, Chia-Te Lin, Ke-Chia Tseng, and Li-Kang Chu* Department of Chemistry, National Tsing Hua University, 101, Sec. 2, Kuang-Fu Rd., Hsinchu 30013, Taiwan.

Corresponding Author *To whom correspondence should be addressed. Phone: 886-3-5715131 ext. 33396. Fax: 886-3-5711082. E-mail: [email protected].

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ABSTRACT: Photoexcitation of SiO2-coated gold nanorods (AuNR@SiO2) with a 1,064-nm pulse of ca. 100 µs duration leads to a temperature jump of 5 oC, as determined by the evolution of the fluorescence intensity of tryptophan. A confocal fluorescent thermometer was developed to detect the temperature evolution in a small excitation volume of ca. 10–3 mm3 and provide a spatial resolution of 100 µm. In addition, a continuous-wave laser at 1,550 nm was employed to increase the initial temperature to 44 oC by heating H2O, providing alternative initial temperatures for temperature jump experiments to reveal the unfolding kinetics of bovine serum albumin (BSA). The evolution of fluorescence of BSA upon temperature jump when the stationary temperature was raised to 44 oC differed from that at room temperature, suggesting a dynamical unfolding kinetics, and a rate coefficient of 75 ± 15 s–1 was derived. In this work, we successfully demonstrated the applicability of AuNR@SiO2 as the photothermal material for the temperature jump and employment of a confocal fluorescent thermometer to precisely monitor the minuscule heating volume. It would be advantageous to utilize this apparatus as an alternative tool for studying the kinetics of protein unfolding.

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1. INTRODUCTION Relaxation methods are widely used in understanding the kinetics of protein unfolding by means of temperature jump (T-jump),1,2,3,4,5,6 pressure jump (P-jump),2,7,8 and pH jump.9 In T-jump experiments, the instantaneous temperature increase can be accomplished by pulsed photoexcitation of H2O,10,11 D2O,10,11 and dyes,12 and by electrical discharge.13 The excitation wavelengths for the overtones of H2O or D2O (Refs. 10,11) are in the middle infrared region (1.5–2.0 µm), which can be generated by a Raman shifter pumped by intense nanosecond Nd:YAG laser at 1,064 nm to provide a temperature increment larger than 5 K.2,10,11 However, several high pressure gases, such as H2 (Refs. 3,14), D2 (Ref. 10), and CH4 (Ref. 15), entail an explosion risk when used as Raman conversion materials. Alternatively, nonlinear optics could be employed to tune the wavelength in the near infrared using the idler beam from an optical parametric oscillator pumped by a second harmonic Nd:YAG laser.16 In this work, gold nanorods (AuNRs) with surface modification by SiO2 were employed as alternative photothermal materials which can be easily excited by Nd:YAG laser without a complicated nonlinear optical arrangement. AuNRs are extensively used in biological applications, such as thermotherapy,17,18 photodynamic treatment,17,19,20 and controllable drug delivery.21,22,23 The excitation of the longitudinal plasmonic band of gold nanorods in the infrared region24 makes it a promising photothermal convertor17,18,21,22,23 because the excitation wavelengths perfectly fit within the optical therapeutic window (650–1350 nm), which is capable of penetrating biological tissues more efficiently than visible light.25 However, surface-unmodified AuNRs are potentially toxic due to the capping materials, e.g., CTAB,26,27 which might lead to the denaturation or aggregation of proteins.28,29 Therefore, the surface modification of AuNRs to reduce their toxicity and enhance their biocompatibility is essential for biological applications.18,30 Among

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them, silica-coated AuNRs (AuNR@SiO2) are suitable for photothermal transducers because SiO2 is biocompatible and has low toxicity.31 Upon the T-jump, the evolution of the structural alteration can be monitored using the fluorescence of tryptophan,2,4,10 vibrational features of amide bands with time-resolved infrared,1 and Raman spectroscopy.6 In this work, we not only utilized AuNRs as an alternative photothermal material but also redesigned the conventional temperature jump system (Ref. 10) by collecting the fluorescence of tryptophan with a confocal configuration. In this configuration, the excitation and probe beams enter the cuvette in the same direction, as shown in Figure 1a, and the spatial resolution can reach 100 µm. In addition, a continuous-wave 1,550 nm laser was introduced to preheat the solution to ∆T=18 oC in the steady state. The advantage of this system is its capability to detect a small heating volume and flexibility for signal optimization. We therefore utilized this approach to investigate the unfolding kinetics of bovine serum albumin (BSA), which contains two tryptophans, Trp-134 and Trp-213,32 to serve as the fluorescent indicator.33 The conformational change is reversible below 50 oC (Refs. 34,35), and the heat-induced unfolding and aggregation of BSA starts at 65 oC.36 Additionally, the presence of AuNR@SiO2 might prevent the potential denaturation of BSA caused by CTAB.28 In this work, a moderate temperature jump of 5 oC was generated from different starting temperatures (below 45 oC) by 1,550 nm optical preheating of H2O. The distinguishable evolutions of fluorescence manifested the dynamical structural alteration attributed to reversible unfolding or unconsolidation of BSA. The successful launch of this approach makes it possible to study the dynamics and kinetics of the proteins as a whole upon thermal stimulus.

2. EXPERIMENTAL SECTION Materials preparations 4


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Synthesis of gold nanorods capped with CTAB (AuNR@CTAB). Seeding gold nanoparticle suspensions were prepared according to the procedure by Vigderman and Zubarev37 with modification. 920 µL of solution containing 0.01 M of sodium borohydride (NaBH4, Sigma-Aldrich, ≥ 98 %) and 0.01 M sodium hydroxide (NaOH, J. T. Baker, ≥ 98.7 %) was freshly prepared under ice bath and denoted as Solution A. After 172 µL of 0.029 M chloroauric acid solution (HAuCl4, Alfa Aesar, ≥ 99.9 %) and 10 mL of 0.1 M cetyltrimethylammonium bromide solution (CTAB, Alfa Aesar, 98 %) were mixed, 920 µL of iced Solution A was then added under rapid stirring until the solution became translucent brown for further use as the seed solution. Growth solution was prepared by mixing 1.376 mL of 0.029 M HAuCl4(aq) with 5.6 mL of 0.01 M AgNO3(aq) (Sigma-Aldrich, ≥ 99.8 %) in 69.744 mL of 0.113 M CTAB(aq), followed by the addition of 2 mL of 0.2 M hydroquinone (Merck-Schuchardt, > 99 %) aqueous solution and shaking till clear. Consequently, 1.28 mL of seed solution was added to the growth solution and kept in a 28 oC water bath to age overnight. The solution was further purified upon centrifugation at 11,515 ×g for 20 minutes and then redistributed with deionized water to 10 mL. This procedure was repeated once, without redistribution the second time, and the solution was kept at room temperature for further surface modification. Synthesis of silica-coated gold nanorods (AuNR@SiO2). The surface coating of SiO2 was applied according to the method by Cong et al.38 with slight modification. AuNR@CTAB solution was adjusted to pH 10.5 by 0.1 M NaOH (J. T. Baker, ≥ 98.7 %), followed by the addition of 400 µL of 25% w.t. tetraethyl orthosilicate (TEOS, Alfa Aesar, 98 %) in ethanol (Sigma-Aldrich, ≥ 99.8 %) with rapid stirring for 30 minutes. Then the solution was aged for 20 hours to allow the formation of SiO2 shells. The solution was centrifuged at 7,370 ×g for

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20 minutes, after which the supernatant was discarded and the concentrate was stored at room temperature for further use. Preparation of mixtures containing tryptophan and BSA in AuNR@SiO2 suspensions. The concentration of AuNR@SiO2 in the mixtures was defined by the absorbance of the longitudinal mode at 1,064 nm (fixed at O.D. = 2.0 for 0.3 cm path length). For preparing the bovine serum albumin (BSA, Sigma-Aldrich, ≥ 98 %) and tryptophan (Sigma-Aldrich, ≥ 99.5 %) mixtures, 75 µL of 80 mg/ml BSA and 75 µL of 0.01 M tryptophan in 10 mM pH 7.0 Tris buffer (UniRegion Bio-Tech, 99.9 %) were mixed with equal volumes of AuNR solution (equivalent O.D = 4.0 at 1,064 nm for 0.3 cm path length) separately, yielding final concentrations of 40 mg/ml BSA and 5 mM tryptophan solution in the presence of AuNR@SiO2. Steady-state spectroscopies Steady-state ultraviolet-visible (UV-Vis) and near-infrared (NIR) absorption spectra were recorded with spectrometers from Ocean Optics (USB4000-UV-VIS) and from JASCO (V-670), respectively. Fluorescence (FL) spectra were recorded with a spectrophotometer from Hitachi (F-7000), and a thermostat from Quantum Northwest (qpod TC125) was mounted on the sample compartment to control the temperatures at 20–55 °C within 0.1 oC variation. The emission contours upon excitation of tryptophan and BSA at 280 nm were collected from 290 to 500 nm. Circular dichroism (CD) spectra were measured with a CD spectrometer (Aviv-410) using a quartz cuvette of 1 mm optical path length. Temporally and spatially resolved fluorescence thermometer The apparatus for monitoring the temporal and spatial evolution of the tryptophan fluorescence intensity change during the photoexcitation of the AuNR@SiO2 is shown in Figure 1a. The probe beam and pump beam were introduced from the same side of the quartz cell (Hellma Analytics, 101.015-QS) to maximize the fluorescence signal and heating 6


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efficiency. A broad band light source from an arc lamp (Newport Corporation, 67005) filtered by a bandpass filter (Edmund Optics, 67813) at 280 nm served as the probe beam. The fundamental generated from the Nd:YAG laser (LOTIS, LS-2134UTF) at 1,064 nm in the long-pulse mode served as the pump beam. Another 1,550 nm continuous-wave laser (Lasever Inc., LSR1550NL) was introduced opposite to the 1,064 nm pump beam to raise the initial temperature upon heating water. The fluorescence of tryptophan was collected by a microspot focusing objective (Thorlabs, LMU-20X-NUV) and filtered by a set of filters (Lambda Research Optics, BG-3-25.4; Lambda Research Optics, SWP-2502B-650; Thorlabs, FGB37-A) to define the wavelengths (335–400 nm) of interest. Then the fluorescence was focused through a 100 µm diameter pinhole (Thorlabs, P100S) by a lens (Thorlabs, LA4306-UV) to achieve the spatial resolution. The aforementioned components were constructed in a cage system (Thorlabs) mounted on an XYZ translation stage (Thorlabs, MT3/M), allowing us to scan the probing volume of interest. The fluorescence photons were detected by a photomultiplier tube (Hamamatsu, R928) and recorded by an oscilloscope (LeCroy, 24MXs-B). The transformation of the fluorescence change to the temperature change is shown in Figure 1b. The temperature evolution, ∆Tt , was derived by Eq. 1 ∆Tt = α-1 × (V0–Vt)/V0

(Eq. 1),

where V0 and Vt denote the voltage outputs from the photomultiplier tube, which are proportional to the tryptophan fluorescence upon 280 nm illumination without and with the 1,064 nm pulsed laser excitation of AuNR@SiO2, respectively. α is ca. 2 % oC–1 for the fluorescence decrement derived from the temperature-dependent fluorescence, as shown in Figure S1. Transmission electron microscope (TEM) imaging

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The morphology of the AuNRs was acquired with a high resolution transmission electron microscope from JEOL Ltd. (JEM-2100). The electron microscopic images of as-prepared and purified silica-coated AuNRs are shown in Figure 2, and those after laser irradiation are shown in Figure S2. 3. RESULTS AND DISCUSSIONS The morphologies and the extinction spectra of the AuNR@SiO2 were characterized with the electron microscopic images and steady-state absorption spectroscopy, respectively. The modulation of the fluorescence intensity of tryptophan upon excitation of the mixtures of tryptophan and AuNR@SiO2 at 1,064 nm reflects the temperature evolution of the solution. An external 1,550 nm c.w. laser was used to preheat H2O to raise the initial temperature. The fluorescence intensity evolution of BSA upon temperature jump reflects the kinetics of a reversible unfolding process. Characterizations of AuNR@SiO2 before pulsed laser excitation The electron microscope images of the as-prepared CTAB-capped gold nanorods, as-prepared SiO2-coated gold nanorods, and purified SiO2-coated gold nanorods (AuNR@SiO2) are shown in Figure 2a–2c, respectively. The morphology of the core AuNRs was not significantly changed after the surface modification of SiO2 with TEOS, in terms of the averaged aspect ratios of 6.4 ± 0.9 and 6.1 ± 1.1, respectively, and the thickness of the SiO2 layer of 13 ± 2 nm. Purification upon centrifugation at 7,370 ×g for 20 minutes did not cause severe aggregation (Figure 2c), and the AuNRs had a similar aspect ratio of 6.3 ± 1.1. This is also revealed in the normalized extinction spectra (Figure 2d). The surface plasmonic bands of longitudinal mode slightly redshifted from 990 nm to 1,025 nm upon changing of the capping material CTAB to SiO2, which is consistent with the previous report by Zhan et al.39 The spectral contours of as-prepared SiO2-coated AuNRs and those after centrifugation were similar, supported by the electron microscope images. Thus, the purification of AuNRs 8


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by centrifugation can efficiently exclude other chemicals in the synthesis which could potentially affect the properties of tryptophan and BSA in subsequent measurements. Temperature evolution upon 1,064-nm photoexcitation of AuNR@SiO2 Previous studies showed that the intensity of the tryptophan fluorescence decreases as the temperature increases.40,41 The fluorescence contours of tryptophan in the presence of AuNR@SiO2 at different temperatures are shown in Figure S1. The normalized contours in that figure are almost the same, indicating that the electronic energetics of tryptophan is not influenced in the presence of AuNR@SiO2 at 25–45 oC. The relationship of the fluorescence intensity decreases and the temperature increment is about 2 % oC–1, consistent with previous reports.41 This observation suggests that the presence of AuNR@SiO2 will not affect the fluorescence property of tryptophan and confirms the applicability of AuNR@SiO2 as photothermal transducers when using tryptophan in fluorescence thermometry. Upon pulsed excitation of the AuNR@SiO2 at 1,064 nm, the temperature changed with time, as shown by the black circles in Figure 3. In order to confirm the origin of the temperature increase, the pulse width of the 1,064 nm excitation laser was recorded (red trace in Figure 3). The normalized accumulated energy of the excitation pulse (blue trace in Figure 3) coincided with the rise in temperature, suggesting that the temperature change could be attributed to photothermal transformation upon excitation of AuNR@SiO2, and the rise time was about 63 µs (10 % to 90 %). The temperature reached the plateau and remained unchanged within 10 ms, sufficiently providing an instantaneous temperature jump and stationary temperature for the further study of protein unfolding kinetics. Spatially-resolved temperature evolutions Since the present fluorescent thermometer was constructed in the confocal configuration, we were able to illustrate the temperature evolution at different probe positions along the z-axis, as shown in Figure 4a. By plotting the temperature at the plateau averaged 9



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for 150–900 µs, the spatial distribution of the temperature, ca. 1.3 mm upon fitting with a Gaussian function, satisfactorily coincided with the diameter of the 1,064 nm laser cross section (about 2 mm), as shown in Figure 4b. This experimental approach is advantageous to the detectability of the small heating volume using focused excitation laser and prevents averaging of the fluorescence signal from the unexcited volume due to the imperfect spatial overlap of the excitation and probe lasers, providing better spatial resolution for the conventional temperature jump apparatus.10 Dependence of the concentrations of AuNR@SiO2 and fluxes of 1,064 nm pulsed laser The temperature reaching the plateau, ∆Tmax, can be expressed by the following equation,

(

)

∆Tmax ∝ ∆I = I 0 × 1 − 10 −O.D./30 ≈ 2.303 × I 0 × O.D./30

(Eq. 2)

when O.D./30 is small. I0 and O.D. denote the energy of the incident laser at 1,064 nm and the optical density of the AuNR@SiO2, respectively. ∆I refers to the number of the 1,064-nm photons absorbed by the AuNR@SiO2 that lead to the temperature increase, ∆T. The effective absorption optical length (100 µm) of the AuNR@SiO2 in the detection volume, which is defined by the diameter of the pinhole in the confocal configuration, was taken into consideration, and a factor of 30 was determined relative to the length of the inner side of the quartz cuvette (3 mm). Accordingly, the ∆Tmax should be linearly proportional to the incident laser energy, I0, and the concentration of AuNR@SiO2, in terms of O.D. The temperature evolutions of AuNR@SiO2 suspensions of different concentrations, in terms of O.D., were recorded upon 1,064 nm excitation (Figure 5a). The plot of O.D. and the maximum temperature difference, ∆Tmax, in 150-900 µs manifested satisfactory linearity (Figure 5b), suggesting that the photons absorbed by the AuNRs were responsible for the increase in temperature. Besides the concentration dependence, the incident excitation fluxes 10


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were adjusted to confirm the linearity and the damage threshold of the AuNR@SiO2. The temperature evolutions upon excitation of AuNR@SiO2 of O.D. = 2.0 using different 1,064-nm laser fluxes are shown in Figure 5c. The plot of the incident 1,064 nm laser flux and ∆Tmax in 150-900 µs manifested satisfactory linearity (Figure 5d), suggesting that the maximum temperature difference can be manually controlled by tuning the incident laser fluxes. After the laser irradiation, the morphologies and extinction spectra of AuNR@SiO2 were also recorded, as shown in Figure S2. Retention of the shapes and extinction spectrum suggested that the AuNR@SiO2 withstood the exposure to 1,064 nm laser of ca. 100-µs pulse width at 1.9 mJ cm–2, i.e., the damage threshold. Raising the stationary temperature with a continuous-wave 1,550 nm laser The combination band of H2O, ν1 (symmetric stretch) + ν3 (asymmetric stretch) or overtone of OH stretch, manifests a moderate infrared strength at 1,450–1,500 nm.6,16,42 We introduced an external 1,550 nm laser to preheat H2O to raise the initial temperature, which can be used for further study of the protein unfolding process. Figure 6a shows the evolutions of temperature from exposing the mixture containing tryptophan and AuNR@SiO2 to 1,064-nm pulsed laser at 0.5 Hz and 1,550-nm continuous-wave laser at different excitation fluxes, simultaneously. When the flux of the incident 1,550-nm laser was about 648 mWatt cm–2 (beam of a diameter of 7.5 mm at 285 mWatt), the temperature gradually increased and reached a steady state of 44 oC, at which the transient temperature jump profiles were collected (blue trace in Figure 6b). This method provides great flexibility in tuning the environment temperature using optical heating. The combination of stationary exposure, pulsed excitations, and a confocal fluorescent thermometer offers a satisfactory approach for studying the protein unfolding kinetics of, e.g., bovine serum albumin (BSA). Reversible unfolding kinetics of bovine serum albumin

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Bovine serum albumin (BSA) is a water-soluble protein, and the helicity is composed of 66% α-helix, 3% β-structure, and 31% random coil in its secondary structure.43 BSA contains two tryptophans, Trp-134 in the first domain and Trp-212 in the second domain.44 Although BSA has 20 tyrosines45 which fluoresce upon UV excitation, the content at λFL > 335 nm, defined by the optical filter in the present fluorescent thermometer apparatus, is ignorable.46 Hence, the evolution of the fluorescence of the tryptophans can serve as the detection window for the thermally-induced structural alteration of BSA. When proteins are integrated with silica-coated metallic nanostructures, the proteins will probably aggregate on the nanoparticle surface to form a protein corona,47,48 which might hamper the detection of the native properties of individual proteins. We collected the UV-Vis absorption and circular dichroism spectra for pure BSA and the supernatant of the mixtures containing BSA and AuNR@SiO2 after centrifugation, as shown in Figure 7a and 7b, respectively. We found a < 2% decrease in the absorbance at 280 nm after centrifugation of the mixture of BSA and AuNR@SiO2. Even assuming that the decrease in absorbance is thoroughly attributable to the protein corona on the AuNR@SiO2, the effect of the protein corona on the fluorescence signal would play an ignorable role. This conclusion is also supported by the high similarity of the CD spectra (Figure 7b). The secondary characteristics of BSA will not change in the presence of AuNR@SiO2. As a result, the protein corona could be excluded in the subsequent temperature jump experiments. BSA will not irreversibly denature at temperatures lower than 50 oC.34,35 When the temperature was increased from room temperature, the fluorescence intensity of BSA gradually attenuated (Figure 8a), attributed to the temperature dependence. Meanwhile, the wavelength maximum shifted hypsochromically with an obvious kink at about 40 oC (Figure 8b), indicating a less flexible micro-environment for the indole ring of tryptophan.49 As a result, raising the initial stationary temperature could lead to a difference in the evolution of 12


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∆IFL(t)/IFL(0) upon T-jump (Figure 9a) due to the structural change. When the starting temperature was 39 oC, the normalized temporal profile was similar to that at 26 oC. However, when the T-jump experiment was performed at 44 oC, the temporal profile was distinct from the other two. It is reasonable that the structure of BSA was dynamically altered at 49 oC, close to the irreversible denaturation temperature, when a jumped temperature of 5 oC was initiated at 44 oC. Since the thermalization of the jumped temperature, T(t), and unfolding kinetics, G(t), contribute to BSA’s apparent profiles of ∆IFL(t)/IFL(0), F(t), F(t) = T(t) ⊗ G(t)

(Eq. 3)

we therefore divided the T-jump profile at 44 oC, F44(t), with respect to that at 26 oC, F26(t), to eliminate the contribution from the thermalization attributed to the jumped temperature by 1,064 nm and to extract the unfolding kinetics at 44 oC, G44(t), as shown in Figure 9b. The CD spectra of BSA before and after the T-jump experiments are shown in Figure S3, and the features are almost identical to that at room temperature, suggesting that the observed unfolding dynamics was not due to the irreversibly denatured BSA. Upon fitting of G44(t) with a single exponential component, a time constant of 75 ± 15 s–1 was obtained. Although we were not able to differentiate the localized structural alteration, since BSA contains two tryptophans, the apparent rate coefficient still reflected the reversible global structural change upon T-jump near the denaturation temperature. This method could be also employed in the DNA melting that occurred within the millisecond time scale.50 Moreover, upon utilizing this approach to study the unfolding processes, the structural stability of the targeted proteins in the presence of AuNR@SiO2 should be examined beforehand to avoid the nanoparticle-induced structural alteration and the incorrect determination of the kinetics parameters.

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By using a confocal fluorescent thermometer and photoexcitation of SiO2-coated gold nanorods (AuNR@SiO2) with a 1,064-nm pulsed laser, we successfully established a spatially and temporally resolved approach for conducting temperature jump experiments. A transient temperature can reach 5 oC and last for 10 ms. Meanwhile, a continuous-wave laser at 1,550 nm was introduced to increase the initial temperature to 44 oC by optically heating H2O, offering alternative starting temperatures for temperature jump experiments. We found that the T-jump evolution of the fluorescence depletion kinetics of BSA at a starting temperature of 44 oC revealed different unfolding kinetics in comparison with that at room temperature, indicating a global structural change. It needs to be emphasized that this promising approach could be an alternative tool for studying protein unfolding kinetics as a whole without partitioning of the proteins of interest. Moreover, by modifying the proteins with other fluorophores, the present optical approach provides great flexibility in detection windows and wide temperature ranges of interest by optical heating of water. ASSOCIATED CONTENT Steady-state fluorescence spectra of tryptophan in the presence of AuNR@SiO2, electron microscopic images of AuNR@SiO2 after pulsed laser irradiation, and circular dichroism spectra of BSA are supplied as Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * To whom correspondence should be addressed. Phone: 886-3-5715131 ext. 33396. Fax: 886-3-5711082. E-mail: [email protected].

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Notes The authors declare no competing financial interests. ACKNOWLEDGMENT The authors are grateful for The Ministry of Science and Technology of Taiwan for this research (MOST 103-2113-M-007-010-MY2 and MOST 105-2113-M-007-014) and the scholarship of College Student Participation in Research Projects for K.C.T. (MOST 105-2815-C-007-009-M). ABBREVIATIONS AuNR@SiO2, silica-coated; AuNR; BSA, bovine serum albumin REFERENCES (1) Davis, C. M.; Dyer, R. B. The Role of Electrostatic Interactions in Folding of β‑ Proteins. J. Am. Chem. Soc. 2016, 138, 1456–1464. (2) Wirth, A. J.; Liu, Y.; Prigozhin, M. B.; Schulten, K.; Gruebele, M. Comparing Fast Pressure Jump and Temperature Jump Protein Folding Experiments and Simulations. J. Am. Chem. Soc. 2015, 137, 7152 −7159. (3) Ding, B.; Hilaire, M. R.; Gai, F. Infrared and Fluorescence Assessment of Protein Dynamics: From Folding to Function. J. Phys. Chem. B 2016, 120, 5103 −5113. (4) Meadows, C. W.; Balakrishnan, G.; Kier, B. L.; Spiro, T. G.; Klinman, J. P. Temperature-Jump Fluorescence Provides Evidence for Fully Reversible Microsecond Dynamics in a Thermophilic Alcohol Dehydrogenase. J. Am. Chem. Soc. 2015, 137, 10060 −10063. (5) Ebbinghaus, S.; Dhar, A.; McDonald, J. D.; Gruebele, M. Protein Folding Stability and Dynamics Imaged in a Living Cell. Nature Methods 2010, 7, 319–323. (6) Huang, C.-Y.; Balakrishnan, G.; Spiro, T. G. Early Events in Apomyoglobin Unfolding Probed by Laser T-jump/UV Resonance Raman Spectroscopy. Biochemistry 2005, 44, 15734 –15742. (7) Liu, Y.; Prigozhin, M. B.; Schulten, K.; Gruebele, M. Observation of Complete Pressure-Jump Protein Refolding in Molecular Dynamics Simulation and Experiment. J. Am. Chem. Soc. 2014, 136, 4265−4272. (8) Prigozhin, M. B.; Liu, Y.; Wirth, A. J.; Kapoor, S.; Winter, R.; Schulten, K.; Gruebele, M. Misplaced Helix Slows Down Ultrafast Pressure-Jump Protein Folding. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 8087–8092. (9) Causgrove, T. P.; Dyer, R. B. Nonequilibrium Protein Folding Dynamics: Laser-Induced pH-Jump Studies of the Helix–Coil Transition. Chem. Phys. 2006, 323, 2–10.

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Figure 1. (a) Layout of the confocal temperature-jump fluorescence thermometer. See details in the texts. (b) The procedure to derive the temperature evolution: (i) the temporal profile of the fluorescence intensity change of tryptophan upon exciting AuNR@SiO2; (ii) the transformation of the temperature evolution using Eq. 1.

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Figure 2. Electron microscopic images of (a) as-prepared CTAB capped gold nanorods, (b) as-prepared silica-coated gold nanorods, and (c) purified silica-coated gold nanorods (AuNR@SiO2) upon centrifugation at 7,370 ×g for 20 minutes before pulsed laser irradiation. The scale bar denotes the length of 50 nm. (d) The normalized extinction spectra of the aforementioned materials.

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Figure 3. Temperature evolution (black circles) upon excitation of AuNR@SiO2 (O.D. = 2.0 with optical length = 3 mm) at 1064-nm with 1.9 J pulse–1 cm–2 operated at 0.5 Hz. The concentrations of tryptophan and tris buffer are both 5 mM. The red trace denotes the temporal profile of the excitation pulse width. The blue trace denotes the normalized accumulated intensity of the excitation pulse with respect to the temperature maximum of the temperature evolution (black circles).

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Figure 4. (a) Two dimensional contour of the temperature evolution upon scanning the probe positions in the vertical direction (z-axis) of the sample cuvette. (b) The relationship of the ∆Tmax, averaged for 150–900 µs, and the probe positions. The concentration of AuNR@SiO2 is O.D. = 2.0 at 1064 nm. The flux of the excitation laser is 1.9 J pulse–1 cm–2. The concentrations of tryptophan and tris buffer are both 5 mM.

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Figure 5. (a) Temperature evolutions upon excitation of AuNR@SiO2 of different concentrations (O.D. for the optical length of 3 mm). The excitation flux is 2.8 J cm–2. (b) The relationship of the AuNR@SiO2 concentration and ∆Tmax averaged for 150–900 µs. (c) Temperature evolution of AuNR@SiO2 upon 1064 nm excitation at different fluxes. The concentration of AuNR@SiO2 is O.D.=2.0 for the optical length of 3 mm at 1064 nm. (d) The relationship of the laser excitation flux and ∆Tmax averaged for 150–900 µs. The concentrations of tryptophan and tris buffer are both 5 mM.

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Figure 6. (a) The profiles of temporal change, ∆T(t), upon raising the initial temperature of the mixture containing tryptophan and AuNR@SiO2 with a 1550 nm continuous-wave laser at different excitation powers. 1064 nm pulsed laser (1.9 J pulse–1 cm–2) was introduced into the cuvette operated at 0.5 Hz. After 8 minutes for the temperature stabilization (ca. 3 oC increment), the 1550 nm laser at different powers was introduced into the cuvette and the temperature gradually reaches stabilization. (b) The temperature jump profiles at the stationary temperature plateau upon simultaneous irradiation of 1550 nm and 1064 nm lasers, as indicated by the yellowish shadow. The concentration of the AuNR@SiO2 is O.D.=2.0 for 3 mm optical length in the presence of 5 mM tryptophan and 5 mM Tris buffer.

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Figure 7. (a) UV-Vis absorption and (b) circular dichroism (CD) spectra of the diluted sample (1/60) which originally contained pure BSA (40 mg/mL) in 5 mM Tris buffer (black trace) and the supernatants of the mixtures containing BSA (40 mg/mL) and AuNR@SiO2 (O.D.=2.0 of 3 cm optical length at 1064 nm) in 5 mM Tris buffer after centrifugation at 3,420 ×g for 1 hour (red trace).

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Figure 8. The steady-state fluorescence of BSA (40 mg/mL) in the presence of AuNR@SiO2 and tris buffer (5 mM) in temperature range of 25–55 oC. The normalized traces are shown in the inset. (b) The wavelength at maximum of the fluorescence at different temperatures.

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Figure 9. (a) The normalized temperature jump (T-jump) profiles, ∆IFL(t)/IFL(0), of mixtures containing AuNR@SiO2 (O.D.=2.0) and BSA (40 mg/mL) in the presence of 5 mM tris buffer at different initial temperatures. The instantaneous temperature increase is ca. 5 oC. (b) The evolution of the ratio of the T-jump profiles at starting temperature of 44 oC with respect to that at room temperature. The curve is fitted with one time constant, 75 ± 15 s–1, in red sloid line.

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Table of Contents Graphic

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Using SiO2-Coated Gold Nanorods as Temperature Jump

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